This research will be done primarily in University of Tucuman, Argentina in collaboration with Raul Salomon as an extension of NIH grant # R01GM64530. The long-term goal is to understand the structure, function and maturation of 21 amino acid antibiotic Microcin J25 (MccJ25). Our previous work showed that MccJ25, which is produced from a 58 amino acid-long McjA precursor by the action of McjB and McjC proteins targets bacterial RNA polymerase. Further research showed that Mccj25 has a unique structural fold--a threaded lasso. The complementary strengths of the US and Argentinian groups will be combined to address the following specific aims: 1. To establish structure-functional relationships in MccJ25 molecule. Systematic site-specific and random mutagenesis of the MccJ25 moiety of the plasmid-born mcjA gene will be performed. The ability of cells beating mutant mcjA plasmids to produce mutant MccJ25 in the cultured medium in the presence of a complementary plasmid bearing mcjBC maturation genes and mcjD immunity gene will be established; MccJ25 mutants will be purified and their antibacterial and transcription inhibitory functions will be established by in vivo and in vitro tests, respectively. MccJ25 mutants active against cells resistant to wild-type MccJ25 will also be sought. 2. To establish an in vitro system for MccJ25 maturation. Purified recombinant McjA precursor and McjB and McjC will be prepared and used to find conditions for efficient maturation of McjA into MccJ25. The results of this work will clarify the details of MccJ25 synthesis, structure-function and interaction with its target and thus will allow new insights into the mechanism of transcription. Understanding of MccJ25 maturation may allow to use McjA-based chimeric proteins to construct novel cyclic peptides and or proteins with potentially interesting biological and pharmacological properties.